The long-term goal of the proposed research is to improve the understanding of the molecular mechanisms by which mutations in genes that regulate rhodopsin trafficking and photoreceptor membrane renewal lead to retinal diseases. Mutations in rhodopsin that affect its targeting motifs cause severe forms of autosomal dominant retinitis pigmentosa (ADRP). In the course of our research, we defined the rhodopsin C-terminal VxPx and FR motifs as the conserved ciliary targeting signals. Furthermore, we identified a ciliary targeting complex that recognizes these signals and regulates sorting into post-Golgi rhodopsin transport carriers (RTCs). Our previous studies revealed that membrane targeting to the rod outer segments (ROS) is a conserved form of ciliary targeting, and that the complex we identified is a conserved complex that targets sensory receptors to primary cilia through intricate functional networks of small GTPases and their regulators that are exquisitely sensitive to mutations causing retinal degenerations and ciliopathies. We now propose to study the mode of activation and order of assembly of the ciliary targeting complex, identify the single remaining unknown SNARE that mediates RTC fusion, and define the point of intersection of trafficking of integral and peripheral ROS membrane proteins that participate in phototransduction. To accomplish this, we will use our established cell-free system that reconstitutes rhodopsin trafficking in vitro and probe molecular interactions by biochemical assays, co-immunoprecipitation, and pulldown experiments with recombinant and purified components-as well as by transgenic expression of mutant and phosphomimetic protein constructs, enzymatic assays, confocal microscopy, and the in situ Proximity Ligation Assay (PLA). The basic understanding of the molecular linkage between the regulatory machineries involved in the renewal of light-sensitive membranes through ciliary targeting and a wide range of systemic cilia disorders is expected to provide the foundation for improved therapeutic strategies to treat retina-specific and syndromic forms of photoreceptor loss.